Antibiogram profiling and detection of icaA and blaZ genes from Staphylococcus aureus and coagulase-negative Staphylococcus spp. of healthy bovine raw milk sample origin

Objective: This study focused on the antibiogram profiling of Staphylococcus aureus and coagulase-negative Staphylococcus spp. (CoNS) and the detection of icaA and blaZ genes from bovine raw milk samples. Materials and Methods: Bovine milk samples were collected from dairy farms, and Staphylococcus spp. were isolated and identified via conventional and molecular screening. Disk diffusion test (DDT) was implemented to determine the resistance pattern. Biofilm and β-lactamase-producing Staphylococcus spp. were identified via amplification of the icaA and blaZ genes. Methicillin-resistant Staphylococcus aureus and CoNS were identified by DDT and PCR of the mecA gene. Results: From 63 samples, 35 were confirmed as Staphylococcus spp., of which 16 (25.39%) S. aureus isolates were coagulase-positive, while 19 (30.16%) were negative. PCR confirmed that 50% (8/16) of S. aureus and 36.84% (7/19) of CoNS possessed the icaA gene. All S. aureus isolates were found resistant to penicillin-G (P) both phenotypically and genotypically. The isolates were also resistant to erythromycin (ERY) and oxytetracycline (TET). While CoNS showed high to reduced resistance against P, TET, ERY, and azithromycin, no S. aureus isolates were resistant to sulfamethoxazole, while 10.53% of CoNS isolates were. All S. aureus and CoNS isolates were susceptible to vancomycin and gentamicin. MR was exhibited by 37.5% of S. aureus and 42.10% of CoNS isolates. Moreover, S. aureus and CoNS had 56.25% and 52.63% multidrug-resistant (MDR) isolates, respectively. Conclusion: The present study revealed the presence of a biofilm-producing, MDR staphylococcal strain in milk that might endanger consumers. Routine surveillance and monitoring, along with antimicrobial resistance learning, can reduce risks.


Introduction
Most mastitis is caused by bacteria; however, viral, algal, and fungal strains have also been reported.The teat canal accommodates bacteria into the bovine mammary gland, causing "mastitis" [1].Nearly 200 infectious agents of mastitis in bovine have been identified till now, with Coliforms, Streptococcus agalactiae, Staphylococcus aureus, and other Streptococci being the most prevalent microbes found in large animals [2].S. aureus is the primary cause of bovine mastitis, the most frequent dairy complication [3].Although mastitis among cattle is induced by plenty of factors, S. aureus is the primary global cause of bovine mastitis [4].S. aureus, a major food-borne pathogen, infects humans and animals [5].Recently, in most countries, coagulase-negative staphylococci (CoNS) elicit intramammary infections (IMIs), despite S. aureus being the most dangerous mastitis-causing pathogen in cattle [6].
Nevertheless, clinical or pathogenic significance while isolated from milk remains controversial; some address mastitis-causing bacteria with significant virulence factors [7].The key pathogenic hallmark of S. aureus and CoNS in mastitis is biofilm formation.[8].Various species have been recorded.Due to the rising role of CoNS in bovine mastitis, species-level CoNS detection is crucial to developing efficient management approaches [9].
Clusters of microorganisms on various surfaces are called "biofilm."Biofilm formation in S. aureus boosts the expansion of antibiotic resistance.Many genes contribute to biofilm formation; however, the icaA gene is a crucial factor for S. aureus strains.The icaA gene produces a transmembrane protein that synthesizes poly-N-acetylglucosamine polymer via N-acetylglucosaminyl transferases [10].
Antibiotic-resistant mastitis-causing bacteria have been reported to result from antibiotic abuse.The diverse therapeutic use of antimicrobials or their frequent use as growth stimulants in animal feed has been associated with human and animal-borne pathogenic microorganism resistance [11].Nowadays, bovine mastitis treatment begins with antibiotics, and the resultants' resistant microbes are rendering antibiotics ineffective.Antibiotic residues additionally jeopardize public health [12].The detection of pathogens in mastitis is crucial for antibiotic selection.β-lactams are extensively utilized in intramammary medication.Anti-β-lactam strategies in bacteria include blaZ gene-encoded β-lactamases and mecA gene-encoded low affinity penicillin-binding protein (PBP2a).The methicillin-resistance (MR) condition prohibits treatment with known β-lactam antibiotics [13].It is predicted that the occurrence of MR in the CoNS is greater than that of S. aureus.The mecA gene is harbored by MR CoNS, which can be horizontally transferred among staphylococci.Moreover, mecA-positive CoNS might act as vectors for spreading newly detected clones of Methicillin-resistant Staphylococcus aureus (MRSA) [14].In Bangladesh, there has been no published research on biofilm-producing CoNS detection in mastitis-infected cows.The current study assessed antibiotic-resistant and icaA genes containing S. aureus and S. epidermidis in raw milk from selected farms.

Sample collection
From the dairy farms of Bangladesh Agricultural University (BAU) (24.7363°N, 90.4245°E) and Nitish Bohumukhi Dairy Farm, Pulbaria, Mymensingh (24.57961°N, 90.0770°E), a total of 63 fresh milk samples (32 from the BAU dairy farm and 31 from Fulbaria) were collected from wholesome lactating cows utilizing sterilized apparatus.About 10 ml of milk was drawn at random from a single quarter.The milk samples were collected without harming the cows following the guidelines set by the ethical committee of the BAU.The research was conducted at the laboratories of the Department of Microbiology and Hygiene, BAU, Mymensingh.

Isolation of Staphylococcus spp.
Test specimens (0.01 ml, milk) had been streaked over 5% sheep blood agar and incubated overnight at 37°C suspected Staphylococcus spp.colonies were sub-cultured on MSA for pure culture.Colony characteristics on MSA, β-hemolytic motifs on blood agar supplemented with sheep blood (5%, v/v), Gram staining properties, catalase, and coagulase assays confirmed the isolates as Staphylococcus spp.Fresh rabbit plasma and 3% hydrogen peroxide were utilized for catalase and coagulase tests.Finally, Staphylococcus spp. was verified by amplification of the nuc gene.

Extraction of bacterial genomic DNA
In a nutshell, for the extraction, simply one colony had to be placed in the distilled water of 100 µl in an Eppendorf tube, thoroughly mixed, and heated for around 10 min.After heating, tubes were placed on ice for cold shock and centrifuged at 10,000 rpm for 10 min at 4°C.The supernatant was separated and used as a DNA template [15].

Amplification of nuc, mecA, icaA, and blaZ genes by PCR
Using a gradient-based thermocycler, simplex PCR was carried out to amplify the nuc and mecA genes and identify MRSA.Genes associated with biofilm (icaA) and β-lactamase production (blaZ) had been amplified individually.Genomic DNA that had tested positive previously for the specified genes was utilized as positive controls.Nontemplate controls were established using PBS instead of genomic DNA for negative controls.The thermal profiles of three PCRs (first PCR: nuc plus mecA; second PCR: icaA; third PCR: blaZ) were initial denaturation at 95°C for 5 min; the sample underwent 30 cycles of denaturation at the same temperature for 1 min; final extension at 72°C for 10 min; and holding at 4°C.The annealing temperatures were 55°C for 45 sec, 50°C for 30 sec, and 46°C for 30 sec to amplify the genes (nuc plus mecA, icaA, and blaZ), respectively.The extension temperatures of these three PCR's were 72°C for 45 sec, 72°C for 1.5 min, and 72°C for 45 sec.The detailed information on primers is presented in Table 1.

Antimicrobial susceptibility testing
Kirby-Bauer's disc diffusion technique (DDT) [18] was applied to determine antimicrobial susceptibility.The results were presented according to CLSI standards [19].Ten antibiotic discs from seven different classes were used for the study.By adding normal saline, each isolate's overnight growth was set to a concentration of 0.5 McFarland.
Bacterial cultures were dispersed on Muller-Hinton agar with sterile cotton buds and then air-dried.The antibiotic discs of each group were placed on the bacterial lawn, which was incubated overnight at 37°C, followed by a recording of the zone of inhibition and analyses of the findings.Finally, findings have been described as susceptible (S) and resistant (R).Ferdous et al. [18] described multidrug-resistant (MDR) isolates as those that resist at least one compound from each of the three antimicrobial classes.

Statistical analysis
The Statistical Package for Social Science (SPSS.v.25, IBM, Chicago, IL) was used to conduct the statistical tests.Through descriptive analysis, the prevalence of various variables was calculated.The chi-square test for relatedness was conducted to find out whether or not the frequencies of resistance genes differed.Additionally, an identical test was done to check the variations in the occurrence of phenotypic antibiotic resistance in relation to the presence of resistance genes.

Detection of biofilm-producing genes
Biofilm producing the icaA gene was detected in both coagulase-positive and CoNS samples by PCR.Out of 35 samples, the overall occurrence of the icaA gene was detected in 42.86% (15/35) isolates, of which 50% S. aureus and 36.84%CoNS isolates were found to be harboring the icaA gene, respectively (Table 2; Fig. 1B).

Antibiogram of Staphylococcus spp
Figure 2 depicts the resistance pattern found in both S. aureus and CoNS.The pattern of antibiotic resistance showed that all the isolates of S. aureus (100%) were found resistant to penicillin-G, 75% to erythromycin (ERY), 68.75% to oxytetracycline (TET), and 37.5% to methicillin.On the other hand, 73.68% of CoNS were resistant to penicillin-G, 63.16% to TET, 57.89% to ERY, 42.10% to methicillin, and 36.84% to azithromycin (AZM).S. aureus isolates had all been sensitive to sulfamethoxazole (SUL), gentamicin, and vancomycin, while all CoNS isolates were responsive to SUL and gentamicin.

Relationship of phenotypic and genotypic antibiotic resistance patterns with biofilm-producing genes
Among 16 isolates of S. aureus, 50% were noticed to be icaA-bearing, of which 6 (37.75%) were phenotypically and genotypically resistant to oxacillin (OXA).Statistical analysis showed that OXA-resistant isolates carried significantly higher amounts of the mecA gene than the icaA and blaZ genes (Table 5).Furthermore, 7 (43.75%)mecA-positive isolates (Fig. 1C) indicate MR, and all are blaZ-positive (Fig. 1D) with resistance to penicillin-G, which are presented in Table 4.However, among the 19 isolates of CoNS, 7 isolates (36.84%) were detected as icaA-bearing, of which all were positive for blaZ (Table 6).Although the total blaZ-positive CoNS isolates were 78.95%, the results revealed that 56.25% and 52.63% isolates of S. aureus and CoNS were recognized as MDR because these isolates exhibited resistance to antibiotics from three or more distinct classes (Table 3).Maximum 5 (31.25%) isolates of S. aureus showed resistance to two antibiotics, and 6.25% isolates showed resistance to 5, 6, and 7 antibiotics, respectively.In the case of CoNS, a maximum of 21.05% of isolates were observed to be resistant to 5 antibiotics (Figs. 3 and 4).For S. aureus, the multiple antibiotic resistance (MAR) indices varied from 0.1 to 0.7, while for CoNS, they were 0.1-0.6.

Discussion
Staphylococcus spp., a prevalent zoonotic microorganism of dietary origin, causes many human and livestock illnesses.Colonization in the mammary glands of dairy cattle causes mastitis and other syndromes that contaminate raw milk.are the leading mastitis pathogens and biofilm producers that concern public health [20].Although Ballah et al. [16] reported the morphological and genotypic features of biofilm-forming S. aureus from Bangladesh for the first time, The continuing study prioritized CoNS and S. aureus to determine their association or differentiation based on phenotypic resistance patterns and the presence or absence of blaZ, mecA, and icaA genes, and noticed S. aureus and CNS at rates of 25.39% and 30.16%, respectively.Previously, Jahan et al. [21] isolated S. aureus from milk, similar to this study but greater than Dai et al. [3] and lower than Atyabi et al. [22] and Hashemi et al. [23].Atyabi et al. [22] found 30.27% and 2.89% prevalence in CoNS and S. aureus, respectively.Whereas, it was revealed that 19.56% of milk was coagulase-positive and 12.53% had CoNS, concerning staphylococci as the most    predominant bacteria [22,23].André et al. [24] reported 73% S. aureus at a dairy manufacturing facility in Goiás, Brazil, among which 75%, 70%, and 66.7% were detected from milk handlers, cheese, and milk samples, respectively.The study uncovered that milk handlers, milking equipment, and mammary glands of healthy dairy cattle may contaminate milk with CoNS and S. aureus causing clinical and subclinical mastitis.Konuku et al. [6] stated that an elevated rate of S. aureus indicates poor milking, transportation, and dissemination.The appropriate heating process before refrigeration may reduce S. aureus risk.Immunocompromised individuals, infants, the elderly, and women with pregnancies are most vulnerable to raw milk's bacteria.The pathogenicity of the Staphylococcus genus is regulated by the ica operon, which encodes the polysaccharide intercellular adhesin (PIA) [25].Using PCR, this study found the icaA gene in S. aureus and CoNS at 50% and 36.84%,respectively, which was lower than Gajewska et al. [26].In those findings Gajewska et al. [26] noted the icaA gene only in CoNS (24.1%) and no icaA gene in S. aureus; however, the current research has detected it in both coagulase-positive and negative strains.A mutation on icaA may cause DNA sequence changes that prevent these genes from being amplified [27].

Ingestion of raw or inadequately boiled milk can cause a staphylococcal infection. Staphylococcus aureus and CoNS
Khairullah et al. [28] isolated 5.15% and 4.22% of MDR S. aureus and CoNS isolates from cow milk and farmers' hands in East Java, Indonesia, which was significantly lower than the present study.Patterns in healthcare infrastructure, regulatory policies, socioeconomic conditions, climate, and geography all contribute to this change.The isolates harboring icaA gene showed multidrug resistance as well as MR, which might indicate a strong biofilm producer.Intrinsically, the biofilm-producing genes stimulate bacteria to express themselves as strong biofilm producers, leading to the development of the MDR strain.Phenotypic biofilm production is extremely controlled by some genes; among these, icaA is the most common gene.The presence of a higher percentage of icaA bearing MDR S. aureus and CoNS in milk might pose a risk to human and animal health.Further investigation can be conducted on the biofilm assay using CRA (Congo Red Agar) and the microtiter plate method, along with molecular detection of other biofilm-forming genes.

Conclusion
Though ubiquitous, staphylococci can cause subclinical and persistent intra-mammary infections in cows through various virulence factors.We tested bovine milk from selected farms for Staphylococcus spp.Phenotypic and molecular characteristics identified the isolates as Staphylococcus spp.In isolated strains, S. epidermidis was more common than S. aureus.Biofilm-forming probability and MR were genotypically checked by PCR.Antibiograms showed susceptibility to penicillin-G, TET, ERY, AZM, and OXA.In summary, S. aureus in raw milk indicates food-borne infections and antibiotic resistance.Regular and rigorous observation and hygiene may reduce the danger.
with different superscripts differ significantly (p < 0.05) within the variable under assessment, NA = Not applied.
with different superscripts differ significantly (p < 0.05) within the variable under assessment, NA = Not applied.

Table 2 .
Occurrence rate of icaA bearing S. aureus and CoNS isolated from milk samples.

Table 3 .
Multidrug resistance pattern of S. aureus and CoNS.

Table 4 .
Occurrence of blaZ, mecA and icaA genes in isolated Staphylococcus spp.

Table 5 .
Association of phenotypic and genotypic resistance pattern in S. aureus.

Table 6 .
Association of phenotypic and genotypic resistance pattern in CoNS.